KR102530865B1 - Self-Assembling Cyclic Peptide-Oligonucleotide Conjugates and Manufacturing Method Thereof - Google Patents

Abstract

The present invention relates to a self-assembling cyclic peptide-oligonucleotide complex, wherein the cyclic peptide-oligonucleotide complex of the present invention is structurally stable in solution and can form a self-assembly having a uniform size and shape. A new manufacturing method capable of efficiently preparing self-assembling cyclic peptide-oligonucleotide complexes, which are heterogeneous building block structures, can be provided.

Description

Self-Assembling Cyclic Peptide-Oligonucleotide Conjugates and Manufacturing Method Thereof

The present invention relates to a self-assembling cyclic peptide-oligonucleotide complex, and more particularly, to a structurally stable self-assembling cyclic peptide-oligonucleotide complex and a method for preparing the same.

Cyclization of molecules has many advantages in the development of drugs and new materials. In particular, self-assembly patterns, glass transition temperatures, and decomposition rates can be controlled through cyclization of polymers and peptides.

The self-assembly of heterogeneous building blocks is difficult to control, but offers several advantages in terms of complexity and versatility. In this respect, self-assembling peptide-oligonucleotide conjugates (POCs) have been attracting attention for the past several years, and have shown various applications for cell culture materials, drug delivery, antisense effects, and functionalization of nanoparticles.

However, there are the following problems in synthesizing self-assembling POCs. First, nucleic acids and peptides show completely different physical and chemical properties (melting temperature, solubility, etc.) depending on the sequence, and there is a problem that their chemical synthesis methods are not compatible with each other. For example, DNA has a problem in that it cannot withstand the strong acidic environment used in solid-state synthesis of peptides. In the second conventional DNA and peptide synthesis process, while the DNA and peptide are connected, the self-assembly characteristics of the peptide must be suppressed, but it is almost impossible to control this, and there are problems in that the yield is significantly lowered if not controlled. Due to the height of the barriers to be overcome as described above, the development of methods for synthesizing POCs is still sluggish.

The synthesis of cyclic POCs was first developed in 1999 by Morr's group. Afterwards, a study of a cyclic POCs library was conducted by Bleczinski to develop a material capable of specifically interacting with the protein surface. The method for synthesizing cyclic POCs can be applied only to limited amino acids and very short DNA that do not have a protecting group on the side chain or have a protecting group that is not weak against acid, and the process is complex and sensitive, so it can be reproduced repeatedly. There are various problems such as difficult, high cost, and low yield.

Patent Document 1. Korean Patent Publication No. 10-2016-0136695

Accordingly, the present invention has been made in view of the above problems, and an object of the present invention is to provide a cyclic peptide-oligonucleotide complex having excellent structural stability and DNA double helix binding ability and a self-assembly comprising the same.

Another object of the present invention is to provide a method capable of producing a large amount of cyclic peptide-oligonucleotide complexes.

Another object of the present invention is to provide a cyclic peptide-oligonucleotide complex prepared according to the above production method and applicable uses thereof.

In order to achieve the above object, the present invention provides a cyclic peptide-oligonucleotide complex represented by Formula 1.

[Formula 1]

In Formula 1,

The Pep is a peptide represented by Formula 2, and the Oligo is an oligonucleotide represented by Formula 3,

[Formula 2]

-[X ₁ ] _a -[X ₂ ] _b -[X ₃ ] _c -[X ₄ ] _d -

[Formula 3]

-Y ₁ -Y ₂ -Y ₃ -Y ₄ -Y ₅ -

In Formulas 2 and 3,

Wherein X ₁ , X ₂ , X ₃ and X ₄ are each independently any one selected from the group consisting of leucine (L), phenylalanine (F), glutamine (Q) and lysine (K), and a to d are 1 or an integer in the range of 4.

Y ₁ , Y ₂ , Y ₃ , Y ₄ and Y ₅ are each independently selected from the group consisting of deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate and deoxycytidine monophosphate. It is any one nucleotide that becomes.

According to an embodiment of the present invention, in Formula 2, X ₁ is lysine (K), leucine (L) or glutamine (Q), and X ₂ is leucine (L), glutamine (Q) or phenylalanine (F ), wherein X ₃ is leucine (L), glutamine (Q) or phenylalanine (F), and X ₄ may be lysine (K), leucine (L) or glutamine (Q).

In addition, the peptide may be represented by any one amino acid sequence selected from SEQ ID NOs: 1 to 4.

In addition, the oligonucleotide may be represented by any one base sequence selected from SEQ ID NOs: 5 to 13.

According to another embodiment of the present invention, the complex may be represented by Formula 4 or Formula 5 below.

[Formula 4]

[Formula 5]

According to another embodiment of the present invention, the cyclic peptide-oligonucleotide complex may form a self-assembly in solution.

In order to achieve the above object, the present invention provides a self-assembly formed by self-assembling a self-assembling cyclic peptide-oligonucleotide complex in a solution.

According to another embodiment of the present invention, the self-assembly may be in the form of spherical micelles or vesicles with an average diameter of 10 to 30 nm.

In order to achieve the above other object, the present invention provides a method for preparing a cyclic peptide-oligonucleotide complex comprising the following steps.

1) preparing a linear peptide-oligonucleotide complex by mixing the oligonucleotide represented by Chemical Formula 6 and the peptide represented by Chemical Formula 7; and

2) cyclization by adding CuSO ₄ , ascorbic acid and butanol to the linear peptide-oligonucleotide complex;

[Formula 6]

[Formula 7]

In Chemical Formulas 6 and 7,

The Pep is a peptide represented by Formula 2, and the Oligo is an oligonucleotide represented by Formula 3,

[Formula 2]

-[X ₁ ] _a -[X ₂ ] _b -[X ₃ ] _c -[X ₄ ] _d -

[Formula 3]

-Y ₁ -Y ₂ -Y ₃ -Y ₄ -Y ₅ -

In Formulas 2 and 3,

Wherein X ₁ , X ₂ , X ₃ and X ₄ are each independently any one selected from the group consisting of leucine (L), phenylalanine (F), glutamine (Q) and lysine (K), and a to d are 1 or an integer in the range of 4.

Y ₁ , Y ₂ , Y ₃ , Y ₄ and Y ₅ are each independently selected from the group consisting of deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate and deoxycytidine monophosphate. It is any one nucleotide that becomes.

The oligonucleotide represented by Formula 6 in step 1) may have a disulfide bond removed from the 3' end through 1-a) treating the oligonucleotide represented by Formula 6 with dithiothreitol (DTT).

According to another embodiment of the present invention, the oligonucleotide represented by Chemical Formula 6 and the peptide represented by Chemical Formula 7 in step 1) may be mixed at a molar ratio of 1:1-4.

According to another embodiment of the present invention, in step 1), an organic solvent may be further included.

According to another embodiment of the present invention, the organic solvent is ethanol, methanol, isopropyl alcohol, butanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile, dichloromethane , It may be any one or more organic solvents selected from the group consisting of ethyl acetate, hexane, diethyl ether, benzene, chloroform and acetone.

According to another embodiment of the present invention, in step 2), the equivalent ratio of the linear peptide-oligonucleotide complex, CuSO ₄ , and ascorbic acid may be 1:2-4:5-7.

According to another embodiment of the present invention, step 2) may be performed under an inert atmosphere.

In order to achieve the above another object, the present invention provides a self-assembling cyclic peptide-oligonucleotide complex prepared by the above preparation method.

The present invention relates to a cyclic peptide-oligonucleotide complex of heterogeneous building blocks using a peptide and an oligonucleotide, a self-assembly comprising the same, and a method for preparing the same. In particular, it is possible to provide a new preparation method capable of efficiently preparing a self-assembling cyclic peptide-oligonucleotide complex having a heterogeneous building block structure. The above preparation method can prepare a structurally stabilized cyclic peptide-oligonucleotide complex at low cost, high yield and high purity.

The cyclic peptide-oligonucleotide complex of the present invention is structurally stable in solution and can form a self-assembly having a uniform size and shape. In addition, the cyclic peptide-oligonucleotide complex of the present invention can provide a specific duplex formation ability to a target material having a complementary sequence through the formation of a stable self-assembly. Therefore, the self-assembly of the present invention can be used as a carrier for stably transporting active substances, and can be variously applied to the treatment or detection of various diseases according to the active substances.

1 is a diagram schematically showing the self-assembly structure and DNA double helix formation structure of LinPOC-1 prepared from Preparation Example 2 according to the present invention.
2 is a diagram schematically showing the self-assembly structure and DNA double helix formation structure of CycPOC-1 prepared from Example 1 according to the present invention.
Figure 3 is a view schematically showing the synthesis process of LinPOC-1 prepared from Preparation Example 2 according to the present invention.
4 is a view schematically showing the synthesis process of CycPOC-1 prepared from Example 1 according to the present invention.
5 is a graph of RP-HPLC results for LinPOC-1 prepared from Preparation Example 2 (red graph) and CycPOC-1 prepared from Example 1 (black graph).
6 is a MALDI-TOF spectrum of LinPOC-1 prepared from Preparation Example 2.
7 is a MALDI-TOF spectrum for CycPOC-1 prepared from Example 1.
8 is a MALDI-TOF spectrum of LinPOC-1 prepared from Preparation Example 2 treated with α-chymotrypsin.
9 is a MALDI-TOF spectrum for CycPOC-1 prepared from Example 1 treated with α-chymotrypsin.
Figure 10 shows the fragment structures of LinPOC-1 prepared from Preparation Example 2 and LinPOC-1 prepared from Preparation Example 2 treated with α-chymotrypsin.
FIG. 11 shows the fragment structures of CycPOC-1 prepared from Example 1 and CycPOC-1 prepared from Example 1 treated with α-chymotrypsin.
12 is a CD spectrum of DNA-1, LinPOC-1 prepared from Preparation Example 2, and CycPOC-1 prepared from Example 1.
13 is a graph showing the results obtained by subtracting the spectrum of the same DNA-1 concentration from the CD spectrum for Pep-1 prepared from Preparation Example 1, LinPOC-1 prepared from Preparation Example 2, and CycPOC-1 prepared from Example 1.
14 is a CD spectrum of DNA-1/DNA-2 (DNA-only-duplex) according to temperature.
15 is a CD spectrum of LinPOC-1/LinPOC-2 (L-duplex) according to temperature.
16 is a CD spectrum of CycPOC-1/CycPOC-2 (C-duplex) according to temperature.
17 shows 254 CD spectra of DNA-1/DNA-2 (DNA-only-duplex), LinPOC-1/LinPOC-2 (L-duplex), and CycPOC-1/CycPOC-2 (C-duplex) by temperature. It is a graph showing CD values in nm.
18 is a result of measuring DNA-1/DNA-2 (DNA-only-duplex), LinPOC-1/LinPOC-2 (L-duplex), and CycPOC-1/CycPOC-2 (C-duplex) by fluorescence analysis. am.
19 is a CD spectrum for LinPOC-2 prepared from Preparation Example 3 according to temperature.
20 is an AFM image of self-assembly of LinPOC-1 prepared from Preparation Example 2.
21 is a self-assembled AFM image of CycPOC-1 prepared from Example 1.
22 is an AFM image of a self-assembled L-duplex.
23 is an AFM image of a self-assembled L-duplex.
24 is an AFM image of a self-assembled C-duplex.
25 is an AFM image of a self-assembled C-duplex.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a self-assembling cyclic peptide-oligonucleotide complex represented by Formula 1. The self-assembling cyclic peptide-oligonucleotide complex of the present invention includes an oligonucleotide and a peptide, and the oligonucleotide and the peptide undergo a thiol-maleimide reaction and a copper-catalyzed azide-alkyne cycloaddition. Since peptides and oligonucleotides bind together, they have the advantage of being able to efficiently prepare and provide cyclic peptide-oligonucleotide complexes without being bound by structure or sequence.

[Formula 1]

In Formula 1,

The Pep is a peptide represented by Formula 2 below, and the Oligo is an oligonucleotide represented by Formula 3 below.

[Formula 2]

-[X ₁ ] _a -[X ₂ ] _b -[X ₃ ] _c -[X ₄ ] _d -

[Formula 3]

-Y ₁ -Y ₂ -Y ₃ -Y ₄ -Y ₅ -

In Formulas 2 and 3,

Wherein X ₁ , X ₂ , X ₃ and X ₄ are each independently any one selected from the group consisting of leucine (L), phenylalanine (F), glutamine (Q) and lysine (K), and a to d are 1 or an integer in the range of 4.

Y ₁ , Y ₂ , Y ₃ , Y ₄ and Y ₅ are each independently selected from the group consisting of deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate and deoxycytidine monophosphate. It is any one nucleotide that becomes.

The self-assembling cyclic peptide-oligonucleotide complex consists of a peptide consisting of 4 to 16, preferably 4 to 10, more preferably 5 to 8, most preferably 7 amino acids; and 5 bases. It may be a complex composed of; oligonucleotides. When the number of amino acids in the peptide is less than the lower limit or exceeds the upper limit, it is highly likely that a cyclic structure cannot be formed or that complex preparation itself becomes difficult.

In Formula 2, X ₁ is lysine (K), leucine (L) or glutamine (Q), X ₂ is leucine (L), glutamine (Q) or phenylalanine (F), and X ₃ is leucine (L), glutamine (Q) or phenylalanine (F), and X ₄ may be lysine (K), leucine (L) or glutamine (Q).

In Formula 2, a, b, c, and d indicate that corresponding amino acids (X ₁ , X ₂ , X ₃ and X ₄ ) are respectively linked to a, b, c, and d units, and specifically amino acids of _X1 This a number means that there are b number of amino acids of X ₂ .

In Formula 2, a, b, c and d may each independently be any one integer from 1 to 4, preferably a and d are each independently any one integer from 2 to 3, and b and c are Each independently may be any one integer from 1 to 2. Most preferably, the sum of a, b, c, and d may be 7 to 9, and for this, when one of a and d is 3, the other may be 2.

The peptide (also referred to as 'Pep') represented by Formula 2 is in the form of a random coil, and may be represented by any one amino acid sequence selected from SEQ ID NOs: 1 to 4, preferably represented by SEQ ID NO: 1. it could be

The oligonucleotide (also referred to as 'oligo') represented by Formula 3 may be represented by any one base sequence selected from SEQ ID NOs: 5 to 13, preferably represented by SEQ ID NO: 5 or 9. .

The self-assembling cyclic peptide-oligonucleotide complex may be represented by Formula 4 or Formula 5 below.

[Formula 4]

[Formula 5]

As the peptide-oligonucleotide complexes were cyclized, the overall molecular conformation was hardly affected, but the self-assembly properties were greatly affected. Specifically, the cyclic peptide-oligonucleotide complex can form a self-assembly in solution, and the self-assembly is characterized in that it is in the form of spherical micelles or vesicles with an average diameter of 10 to 30 nm (FIG. 2). At this time, since the cyclic peptide-oligonucleotide complex has a property of forming a double helix with a nucleic acid molecule comprising a sequence complementary to the oligonucleotide of the present invention, it can specifically bind to a nucleic acid molecule having a complementary sequence. can

On the other hand, when the peptide-oligonucleotide complex is linear without cyclization, it shows a heterogeneous state in which fibrous cylindrical micelles and spherical micelles are mixed (FIG. 1).

Since the self-assembly can support and deliver a biologically active substance inside the micelle or vesicle, it can be used as a delivery vehicle, and it has the ability to form a specific double helix for a nucleic acid molecule complementary to an oligonucleotide, so that a specific nucleic acid It can be variously applied as a composition for diagnosis or treatment or prevention of molecules.

One aspect of the present invention relates to a self-assembly formed by self-assembling a self-assembling cyclic peptide-oligonucleotide complex in a solution. The self-assembly is in the form of spherical micelles or vesicles with an average diameter of 10 to 30 nm, is formed by self-assembly through the interaction of the cyclic peptide-oligonucleotide complex, and is present in the cyclic peptide-oligonucleotide complex. By appropriately selecting peptides and oligonucleotides to be used, it is possible to provide self-assemblies with new structures having various functions and effects. In addition, when peptides and oligonucleotides are designed with a specific sequence complementary to a target substance, a self-assembly having high selectivity for the target substance can be obtained.

Therefore, by using the principle of the self-assembly of the present invention, it is possible to manufacture and provide a self-assembly having various interactions, and use it as a carrier or a composition or carrier for diagnosis, treatment, or prevention.

Another aspect of the present invention relates to a method for preparing a cyclic peptide-oligonucleotide complex comprising the following steps.

1) preparing a linear peptide-oligonucleotide complex by mixing a nucleic acid represented by Chemical Formula 6 with a peptide represented by Chemical Formula 7; and

2) cyclization by adding CuSO ₄ , ascorbic acid and butanol to the linear peptide-oligonucleotide complex;

[Formula 6]

[Formula 7]

In Chemical Formulas 6 and 7,

The Pep is a peptide represented by Chemical Formula 2, and the Oligo is an oligonucleotide represented by Chemical Formula 3.

[Formula 2]

-[X ₁ ] _a -[X ₂ ] _b -[X ₃ ] _c -[X ₄ ] _d -

[Formula 3]

-Y ₁ -Y ₂ -Y ₃ -Y ₄ -Y ₅ -

In Formulas 2 and 3,

Wherein X ₁ , X ₂ , X ₃ and X ₄ are each independently any one selected from the group consisting of leucine (L), phenylalanine (F), glutamine (Q) and lysine (K), and a to d are 1 or an integer in the range of 4.

Y ₁ , Y ₂ , Y ₃ , Y ₄ and Y ₅ are each independently selected from the group consisting of deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate and deoxycytidine monophosphate. It is any one nucleotide that becomes.

In Formula 2, X ₁ is lysine (K), leucine (L) or glutamine (Q), X ₂ is leucine (L), glutamine (Q) or phenylalanine (F), and X ₃ is leucine (L), glutamine (Q) or phenylalanine (F), and X ₄ may be lysine (K), leucine (L) or glutamine (Q).

In Formula 2, a, b, c, and d indicate that corresponding amino acids (X ₁ , X ₂ , X ₃ and X ₄ ) are respectively a, b, c, and d bonded, and specifically X ₁ It means that there are a number of amino acids and b number of amino acids of X ₂ .

In Formula 2, a, b, c and d may each independently be any one integer from 1 to 4, preferably a and d are each independently any one integer from 2 to 3, and b and c are Each independently may be any one integer from 1 to 2. Most preferably, the sum of a, b, c, and d may be 7 to 9, and for this, when one of a and d is 3, the other may be 2.

The peptide represented by Formula 2 (also referred to as 'Pep') may be represented by any one amino acid sequence selected from SEQ ID NOs: 1 to 4, preferably represented by SEQ ID NO: 1.

The oligonucleotide (also referred to as 'oligo') represented by Formula 3 may be represented by any one nucleotide sequence selected from SEQ ID NOs: 5 to 13, preferably represented by SEQ ID NO: 5 or 9 .

A method for preparing the cyclic peptide-oligonucleotide complex will be described in more detail with reference to FIGS. 3 and 4 .

First, 1) a linear peptide-oligonucleotide complex is prepared by mixing an oligonucleotide represented by Chemical Formula 6 and a peptide represented by Chemical Formula 7 (FIG. 3).

In this case, the oligonucleotide represented by Chemical Formula 6 in step 1) may have a disulfide bond removed from the 3' end through 1-a) treating the oligonucleotide represented by Chemical Formula 6 with a reducing agent.

The reducing agent is not particularly limited as long as it can cleave or remove disulfide bonds, but is preferably any one or more reducing agents selected from the group consisting of DTT (dithiothreitol), 2-Mercaptoethanol and TCEP (Tris (2-carboxyethyl) phosphine) It may be the first, and more preferably, it may be DTT (dithiothreitol).

Through the above step 1-a), by cutting or removing the disulfide bond, it is possible to have a free thiol group capable of binding to the maleimide group of the peptide represented by Chemical Formula 7.

Step 1-a) may be performed at 1 to 50 °C for 10 to 120 minutes, preferably at 10 to 30 °C for 30 to 90 minutes.

In addition, the nucleic acid represented by Chemical Formula 6 and the peptide represented by Chemical Formula 7 in step 1) may be mixed in a molar ratio of 1:1 to 4, preferably 1:1.5 to 2.5 molar ratio. If it is less than the lower limit, the binding efficiency of DNA and peptide may decrease, and if it exceeds the upper limit, the self-assembly tendency of peptides becomes too large, resulting in random aggregation, resulting in a decrease in yield.

In step 1), an organic solvent may be further included. The organic solvent is not particularly limited as long as it increases the solubility of the peptide represented by Formula 7 and does not affect the reaction, but is preferably ethanol, methanol, The group consisting of isopropyl alcohol, butanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile, dichloromethane, ethyl acetate, hexane, diethyl ether, benzene, chloroform and acetone It may be any one or more organic solvents selected from.

A linear peptide-oligonucleotide complex is prepared through step 1), and then cyclized by adding CuSO ₄ , ascorbic acid and butanol to the linear peptide-oligonucleotide complex, thereby preparing a cyclic peptide-oligonucleotide complex. do.

In step 2), the equivalent ratio of the linear peptide-oligonucleotide complex, CuSO ₄ , and ascorbic acid may be 1:2-4:5-7.

Step 2) is preferably performed under an inert atmosphere.

Finally, 3) isolating and purifying the cyclic peptide-oligonucleotide complex synthesized in step 2); may be further included.

The separation and purification is not particularly limited as long as it is a peptide separation and purification method, but preferably column chromatography filled with various synthetic resins such as silica gel or activated alumina, filtration and High performance liquid chromatography (HPLC) can be used alone or in parallel, more preferably ultrafiltration, cation exchange chromatography and RP-HPLC (Reverse phase-high performance liquid chromatography) Any one or more separation and purification methods selected from the group consisting of may be used, most preferably RP-HPLC (Reverse phase-high performance chromatography liquid) (eg, C4 reverse phase (RP), C3 RP). there is. The RP-HPLC may be a method of eluting with a mobile phase containing an ion-pair reagent, and the ion-pair reagent uses acetonitrile or triethylammonium acetate (TEAA) to control peak shape and retention time. can be used The ion-pairing reagent may include 0.01 to 1 M triethylammonium acetic acid in distilled water.

Through the above process, the oligonucleotide reacts with the peptide to form a complex, thereby improving the stability of the oligonucleotide that is easily damaged. In other words, the oligonucleotide can be protected by combining the oligonucleotide and the peptide to form a complex having a cyclic structure. Moreover, the cyclic peptide-oligonucleotide complexes can spontaneously assemble to form self-assemblies.

Through the above-described process, a self-assembling cyclic peptide-oligonucleotide complex was synthesized through a simple process using a thiol-maleimide reaction and a copper-catalyzed azide-alkyne cycloaddition (CuAAC). , can be provided with low cost, high yield and high purity. Specifically, the method for preparing the self-assembling cyclic peptide-oligonucleotide complex of the present invention has a yield of 90% or more, and the cyclic peptide-oligonucleotide complex (CycPOC-1) of the present invention requires one purification step (HPLC). It has the advantage of being able to achieve a high yield of 90% or more.

Another aspect of the present invention relates to self-assembling cyclic peptide-oligonucleotide complexes prepared.

The cyclic peptide-oligonucleotide complex is a self-assembling cyclic peptide-oligonucleotide complex represented by Formula 1, and since the description thereof is substantially the same as that described above, redundant description will be omitted.

[Formula 1]

In Formula 1,

The Pep is a peptide represented by Formula 2 below, and the Oligo is an oligonucleotide represented by Formula 3 below.

[Formula 2]

-[X ₁ ] _a -[X ₂ ] _b -[X ₃ ] _c -[X ₄ ] _d -

[Formula 3]

-Y ₁ -Y ₂ -Y ₃ -Y ₄ -Y ₅ -

In Formulas 2 and 3,

Wherein X ₁ , X ₂ , X ₃ and X ₄ are each independently any one selected from the group consisting of leucine (L), phenylalanine (F), glutamine (Q) and lysine (K), and a to d are 1 or an integer in the range of 4.

Y ₁ , Y ₂ , Y ₃ , Y ₄ and Y ₅ are each independently selected from the group consisting of deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate and deoxycytidine monophosphate. It is any one nucleotide that becomes.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are intended to explain the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

experimental material

General Chemicals and Rink amide resin LL were purchased from Merck (Germany) and Fisher Scientific (USA). All amino acids and coupling reagents were purchased from AAPPTec (USA), and DNA (5'-azide-3'-thiol-modified) was synthesized by request from Bioneer (Korea).

Preparation Example 1. Peptide (Pep-1) Synthesis

The peptide LLLFQQKK(Dde) (SEQ ID NO: 1) was synthesized according to a standard solid phase peptide synthesis protocol. The Fmoc (Fluorenylmethoxycarbonyl) protecting group was deprotected by treatment with a 20% piperidine-dissolved N-methyl-2-pyrrolidone (NMP) solution for 15 minutes. All amino acids are HCTU (O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) 4.5 equiv (relative to the resin) and HOBt (1-hydroxybenzotriazole hydrate) 4.5 equiv and DIPEA (diisopropylethylamine) was coupled for 20 minutes using 10 equiv. Amino acids were used after pre-activation 5 minutes before coupling. After coupling the last amino acid, the Dde (1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl) protecting group was added to the peptide with the Fmoc protecting group attached to the peptide, 1 M hydroxylamine and 0.75 NMP solution (3 ml) in which M imidazole was dissolved was added, and the mixture was stirred at room temperature overnight to desorb. The resin was washed with NMP and MC (methylene chloride), and then propionic acid (5 equiv) coupling was performed for 3 hours using an MC coupling reagent in which diisopropylcarbodiimide (DIC) (10 equiv) was dissolved. . After washing the resin again with MC and NMP solutions, deprotecting the final Fmoc protecting group, N-methoxycarbonyl maleimide (5 equiv) and DIPEA (N,N-Diisopropylethylamine) (10 equiv) ) was added to the resin and reacted overnight. After the reaction was complete, the resin was washed with THF and NMP and dried. In order to remove the protecting group remaining on the side chain and separate the peptide from the resin, a cleavage cocktail (TFA:distilled water:TIS=95:2.5:2.5) was added and reacted for 3 hours. After the reaction was completed, the peptide was precipitated using TBME (tert-butyl methyl ether) and purified through RP-HPLC (Waters, USA) using a C4 column.

Preparation Example 2. Preparation of linear peptide-oligonucleotide complex (LinPOC-1)

[Scheme 1]

DNA-1 is represented by GCGAG (SEQ ID NO: 5), and was prepared by ordering from a synthesis company in a state in which an azide group is formed at 5' and a disulfide bond is formed at 3'. The DNA-1 (100 nmol) was dispersed in distilled water and treated with 20 μl of a mixed solution (pH 7) of 0.5 M DTT (dithiothreitol) and 120 mM KPB to remove the disulfide bond at the 3' end of DNA-1. It was reacted for 1 hour. Thereafter, DTT was removed through EA (ethylacetate) extraction. A mixture was prepared by putting the peptide powder (2 equiv, 200 nmol) prepared in Preparation Example 1 into a DNA solution. Acetonitrile (80 μl) was added to increase the solubility of the peptides in the mixture. After stirring the mixture overnight at ambient temperature, RP-HPLC was performed with a semipreparative C4 column using TEAA as an ion paring reagent to separate the desired reactant and MALDI-TOF It was verified by MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry).

Preparation Example 3. Preparation of linear peptide-oligonucleotide complex (LinPOC-2)

DNA is represented by CTCGC (SEQ ID NO: 9), with an azide group at 5' and a disulfide bond at 3'. A linear peptide-oligonucleotide complex (LinPOC-2) was prepared.

Example 1. Preparation of cyclic peptide-oligonucleotide complex (CycPOC-1)

[Scheme 2]

For cyclization, LinPOC-1 prepared from Preparation Example 2 without purification was used.

Copper sulfate pentahydrate (3 μL in 100 mM stock solution) 3 equiv, ascorbic acid 6 equiv (6 μL in 100 mM stock solution) and LinPOC-1 (100 nmol) prepared from Preparation Example 2 without purification 400 μL of solvent (120 μL of water, 80 μL of MeCN, 200 μL of t-butanol) was added. At this time, the reaction was performed in an inert atmosphere to prevent DNA degradation by radicals, and O ₂ was removed using argon gas (Ar) for the inert atmosphere.

The mixture was then left overnight at ambient temperature. The reaction mixture was diluted with 4 mL of a 0.1 M TEAA (triethylammonium acetate) solution. To purify the cyclic peptide-oligonucleotide complex (CycPOC-1) from the mixture, RP-HPLC was performed using 0.1 M TEAA as an ion pairing reagent to isolate the desired product, which was verified by MALDI-TOF MS. As a result, it was confirmed that the yield was 90% or more. It can be seen that the cyclic peptide-oligonucleotide complex (CycPOC-1) of the present invention achieves a high yield of 90% or more through one purification process (HPLC).

Example 2. Preparation of cyclic peptide-oligonucleotide complex (CycPOC-2)

A cyclic peptide-oligonucleotide complex (CycPOC-2) was prepared in the same manner as in Example 1, except that LinPOC-2 prepared in Preparation Example 3 was used instead of LinPOC-1 prepared in Preparation Example 2.

Experimental Example 1. Verification of LinPOC-1 and CycPOC-1 Cyclization

The presence or absence of LinPOC-1 prepared in Preparation Example 2 and CycPOC-1 prepared in Example 1 were verified by RP-HPLC and MALDI-TOF. In addition, in order to confirm the presence or absence of cyclization, the cells were treated with α-chymotrypsin, a sequence-specific enzyme, and then analyzed by MALDI-TOF. The cyclization confirmation process is specifically as follows. An α-chymotrypsin solution dissolved in water at a concentration of 10 mg/ml was prepared, and the concentration of the α-chymotrypsin solution in the LinPOC-1 prepared in Preparation Example 2 and CycPOC-1 prepared in Example 1 solution. was added to 1 mg/ml, allowed to react overnight, and then analyzed by MALDI-TOF.

5 is a graph of RP-HPLC results for LinPOC-1 prepared from Preparation Example 2 (red graph) and CycPOC-1 prepared from Example 1 (black graph).

As shown in FIG. 5, it can be seen that the LinPOC-1 prepared in Preparation Example 2 forms a complex by efficiently conjugating the peptide and the oligonucleotide. The peptide represented by Formula 2 of the present invention has high hydrophobicity and self-assembly characteristics, so it has low solubility in aqueous solution. It can be seen that the problem of poor solubility can be overcome through the conjugation reaction of nucleotides. As the organic solvent, acetonitrile (MeCN) is most preferred.

In addition, a linear POC was formed through a thiol-maleimide reaction, followed by a cycloaddition reaction by mixing a Cu(II) catalyst, ascorbic acid, and t-butanol to prepare a cyclic POC. This can be confirmed through FIG. 5 .

Specifically, the retention time (T _R = 32.1 min) of LinPOC-1 (red graph) prepared from Preparation Example 2 and the retention time (retention time) of CycPOC-1 (black graph) prepared from Example 1 (T _R = 31.4 min) are different from each other, and this difference in retention time means that the overall shape is different. That is, it can be seen that the linear structure was successfully changed to the cyclic structure.

Figure 6 is a MALDI-TOF spectrum for LinPOC-1 prepared from Preparation Example 2, Figure 7 is a MALDI-TOF spectrum for CycPOC-1 prepared from Example 1, Figure 8 is a preparation treated with α-chymotrypsin MALDI-TOF spectrum for LinPOC-1 prepared from Example 2, and FIG. 9 is a MALDI-TOF spectrum for CycPOC-1 prepared from Example 1 treated with α-chymotrypsin. In the drawing, the matrix is 3-hydroxypicolinic acid (3-HPA).

According to Figures 6 and 7, it was confirmed that LinPOC-1 (3195 Da) prepared from Preparation Example 2 and CycPOC-1 (3192 Da) prepared from Example 1 had the same molecular weight. However, in the case of retention time, since it sometimes has an error, another analysis was performed using α-chymotrypsin, which cleaves the peptide bond at the C-terminus of an aromatic amino acid, for more accurate measurement.

8 and 9, it was confirmed that LinPOC-1 prepared from Preparation Example 2 treated with α-chymotrypsin and CycPOC-1 prepared from Example 1 were different in MALDI-TOF spectra. It was confirmed that the predicted structure and molecular weight of each fragment obtained from LinPOC-1 prepared from Preparation Example 2 treated with α-chymotrypsin and CycPOC-1 prepared from Example 1 were consistent with the results of the MALDI-TOF spectrum. .

Comparing the MALDI-TOF spectra of LinPOC-1 prepared from Preparation Example 2 treated with α-chymotrypsin and CycPOC-1 prepared from Example 1, it can be seen that there is no peak in common with each other. That is, it can be seen that CycPOC-1 prepared in Example 1 successfully formed a cyclized structure.

Figure 10 shows the fragment structure of LinPOC-1 prepared from Preparation Example 2 and LinPOC-1 prepared from Preparation Example 2 treated with α-chymotrypsin, and Figure 11 shows CycPOC-1 prepared from Example 1 and The fragment structure of CycPOC-1 prepared from Example 1 treated with α-chymotrypsin is shown. According to this, it can be seen that the structures of the fragments obtained through the treatment of α-chymotrypsin are different due to the difference between the linear and cyclic structures, which was also confirmed through the MALDI-TOF spectrum results (different peaks). That is, it can be seen that CycPOC-1 prepared in Example 1 successfully formed a cyclic structure.

Experimental Example 2. Analysis of the three-dimensional structure of LinPOC-1 and CycPOC-1

CD spectroscopy was used to analyze the three-dimensional structures of LinPOC-1 and CycPOC-1. The CD spectroscopy was performed using a Chiralscan circular dichroism spectrometer (Applied Photophysics., Ltd, UK) equipped with a Peltier temperature controller. CD spectra were recorded from 190 nm to 300 nm using a cuvette with a 2 mm pathlength. At this time, LinPOC-1 and CycPOC-1 were dissolved in distilled water at 20 °C (20 μM, 250 μl), and then analyzed by CD spectroscopy.

12 is a CD spectrum of DNA-1, LinPOC-1 prepared from Preparation Example 2, and CycPOC-1 prepared from Example 1.

According to FIG. 12, the overall CD spectra of LinPOC-1 prepared from Preparation Example 2 and CycPOC-1 prepared from Example 1 showed similar patterns. However, it was confirmed that the minimum value of LinPOC-1 (204 nm) prepared from Preparation Example 2 and CycPOC-1 (202 nm) prepared from Example 1 showed a slight difference. This difference is consistent with the HPLC results observed previously.

13 is a graph showing the results obtained by subtracting the spectrum of the same DNA-1 concentration from the CD spectrum for Pep-1 prepared from Preparation Example 1, LinPOC-1 prepared from Preparation Example 2, and CycPOC-1 prepared from Example 1. The same solvent and concentration were used for each sample (water, 20 μM).

Since cyclization can often change the conformation of a peptide, we wanted to compare the CD spectra for the peptides. To this end, the contribution of DNA was removed from the CD spectra for LinPOC-1 prepared from Preparation Example 2 and CycPOC-1 prepared from Example 1, and the conformation of only the peptide was observed. As shown in FIG. 13, the stereostructures of the peptides in LinPOC-1 prepared from Preparation Example 2 and CycPOC-1 prepared from Example 1 are considered to be the same in the form of random coils, that is, cyclization does not affect the peptide shape at all. confirmed to be

Experimental Example 3. Analysis of DNA duplex formation capability of LinPOC-1 and CycPOC-1

The DNA duplex formation ability of LinPOC-1 prepared from Preparation Example 2 and CycPOC-1 prepared from Example 1 by Watson-Crick base pairing was evaluated using CD spectroscopy and fluorescence spectroscopy.

Specifically, LinPOC-2 of Preparation Example 3 and CycPOC-2 of Example 2 were prepared using DNA-1 (SEQ ID NO: 5: GCGAG) and complementary DNA-2 (SEQ ID NO: 9: CTCGC). Mutually complementary counterparts (DNA-1 and DNA-2, LinPOC-1 and LinPOC-2, and CycPOC-1 and CycPOC-2) were mixed in equal molar amounts in a buffer, DNA-only-duplex, Sufficient reaction time was provided to form L-duplex (LinPOC-1/LinPOC-2) and C-duplex (CycPOC-1/CycPOC-2).

In order to analyze the CD spectrum change with temperature, each of the counterparts (DNA-1 and DNA-2, LinPOC-1 and LinPOC-2, and CycPOC-1 and CycPOC-2) mixed in equal molar amounts was prepared, and Mixed in 300 μl of mM Tris (150 mM NaCl, 10 mM MgCl2, pH 8). The mixture was then incubated overnight prior to CD irradiation, and the CD spectrum was measured while increasing the temperature by 1 °C per minute from 4 °C to 54 °C.

Fluorescence analysis was performed as follows. A sample was prepared under the same conditions as the CD spectrum, EtBr dissolved in water was added to the sample to a final concentration of 20 μM, and the mixture was left for 1 hour before fluorescence measurement, and then the fluorescence spectrum was measured. The excitation wavelength was 520 nm, and the fluorescence spectrum was analyzed from 550 nm to 750 nm.

Changes in the conformational structure of DNA appear very sensitively on the CD spectrum. In addition, the structure of the DNA duplex is unwound into a single strand as the temperature increases, which causes a change in the CD spectrum. Therefore, we tried to analyze the degree of DNA duplex formation through the change of the CD spectrum result according to the temperature change. Specifically, if the DNA duplex is completely formed, the CD spectrum will change significantly as the temperature increases, and if the DNA duplex is partially formed, the CD spectrum will not change significantly.

14 is a CD spectrum of DNA-1/DNA-2 (DNA-only-duplex) according to temperature, according to which, DNA-only-duplex formed by mixing DNA-1 and DNA-2 was confirmed to be a typical B-structure with negative bands at 208 nm and 254 nm and positive bands at 280 nm.

15 is a CD spectrum of LinPOC-1/LinPOC-2 (L-duplex) according to temperature, and FIG. 16 is a CD spectrum of CycPOC-1/CycPOC-2 (C-duplex) according to temperature. 15 and 16, CD spectra for LinPOC-1/LinPOC-2 (L-duplex) and CycPOC-1/CycPOC-2 (C-duplex) are almost identical to those of DNA-only-duplex. It showed the same minimum and maximum values. That is, it can be seen that LinPOC-1/LinPOC-2 (L-duplex) and CycPOC-1/CycPOC-2 (C-duplex) have B-forms.

However, as the temperature increased, the CD spectrum (240 nm ~ 300 nm) of CycPOC-1/CycPOC-2 (C-duplex) showed a low change, but LinPOC-1/LinPOC-2 (L-duplex) The CD spectrum of showed a large change.

17 shows 254 CD spectra of DNA-1/DNA-2 (DNA-only-duplex), LinPOC-1/LinPOC-2 (L-duplex), and CycPOC-1/CycPOC-2 (C-duplex) by temperature. A graph showing the CD value at nm, according to which the change in CD value at 254 nm for C-duplex was lower than that for L-duplex with temperature change. Through this, it can be seen that C-duplex forms significantly fewer DNA double helices than L-duplex.

18 is a result of measuring DNA-1/DNA-2 (DNA-only-duplex), LinPOC-1/LinPOC-2 (L-duplex), and CycPOC-1/CycPOC-2 (C-duplex) by fluorescence analysis. According to this, it was confirmed that less fluorescence of EtBr was observed in C-duplex than in L-duplex. This is consistent with the results of FIG. 17 .

Since the fluorescence intensity of FIG. 18 dramatically increases when EtBr is inserted between DNA base pairs, the degree of formation of a DNA duplex can be seen through the fluorescence intensity. Specifically, the fluorescence intensity of EtBr increases as the degree of formation of the DNA double helix increases, and the fluorescence intensity of EtBr decreases as the degree of formation of the DNA duplex decreases.

As it was confirmed that the fluorescence intensity of C-duplex was lower than that of L-duplex, C-duplex was slightly lower than L-duplex, but it could be seen that it had excellent DNA duplex formation characteristics.

For more accurate comparison, the fluorescence intensity of LinPOC-1 prepared from Preparation Example 2, which is single-straned POCs, was measured. As a result, the fluorescence intensity of EtBr was not observed in the single-stranded LinPOC-1 prepared from Preparation Example 2. It was once again verified that the degree of DNA double helix formation could be confirmed through the fluorescence intensity of EtBr.

19 is a CD spectrum for LinPOC-2 prepared from Preparation Example 3 according to temperature. The LinPOC-2 sample was dissolved in 10 mM Tris buffer (pH 8, 150 mM NaCl, 10 mM MgCl2). The concentration of LinPOC-2 is 15 μM.

As shown in FIG. 19, when only LinPOC-2 is present, it can be seen that no change in the CD spectrum with increasing temperature is observed.

Experimental Example 4. LinPOC-1, CycPOC-1 self-assembly analysis

LinPOC-1 prepared from Preparation Example 2 and CycPOC-1 prepared from Example 1 were photographed with an atomic force microscope (AFM) to confirm self-assembly. First, LinPOC-1 prepared from Preparation Example 2 and CycPOC-1 prepared from Example 1 were each dissolved in water at a concentration of 30 μM, treated with ultrasonic waves, and then allowed to self-assemble overnight. Before AFM analysis, LinPOC-1 and CycPOC-1 were each diluted to a final concentration of 5 μM. LinPOC-1 and CycPOC-1 were loaded on freshly cut mica and dried. AFM images were taken in non-contact mode.

20 is an AFM image of self-assembly of LinPOC-1 prepared from Preparation Example 2, and FIG. 21 is an AFM image of self-assembly of CycPOC-1 prepared from Example 1.

According to FIG. 20, it can be seen that the self-assembly of LinPOC-1 prepared in Preparation Example 2 is formed in a mixture of spherical or fibrous shapes. The average diameter of the spherical self-assembly and the fibrous self-assembly was similar to about 20 nm. Considering that the monomer average diameter of LinPOC-1 prepared from Preparation Example 2 is about 10 nm, it is considered that the spherical self-assembly is in the form of micelles and the fibrous self-assembly is in the form of cylindrical micelles.

According to FIG. 21, it was confirmed that only spherical self-assemblies were homogeneously formed in the self-assembly of CycPOC-1 prepared in Example 1. Its average diameter is about 22 nm, slightly larger than the self-assembly of LinPOC-1.

Experimental Example 5. Formation of double helix self-assembly of LinPOC-1 and CycPOC-1

LinPOC-1 prepared from Preparation Example 2 and CycPOC-1 prepared from Example 1 were photographed with an atomic force microscope (AFM) to confirm self-assembly. First, LinPOC-1 prepared from Preparation Example 2 and CycPOC-1 prepared from Example 1 were each dissolved in distilled water at a concentration of 30 μM, subjected to ultrasonic treatment, and left overnight to self-assemble. Before AFM analysis, LinPOC-1 and CycPOC-1 were each diluted to a final concentration of 5 μM. LinPOC-1 and CycPOC-1 were loaded on freshly cut mica and dried.

In order to confirm the duplex, LinPOC-2 (Preparation Example 1) complementary to independent solutions containing the self-assembly of LinPOC-1 (Preparation Example 1) and the self-assembly of CycPOC-1 (Example 1) 2) and CycPOC-2 (Example 2) were mixed in equal molar amounts, and the mixture was incubated overnight to obtain L-duplex (LinPOC-1/LinPOC-2) and C-duplex (CycPOC-1/CycPOC- 2) was prepared. 5 μl of each of the L-duplex and the C-duplex was taken, loaded onto freshly cut mica, dried, and AFM images were taken in a non-contact mode.

22 and 23 are AFM images of self-assembled L-duplex, and FIGS. 24 and 25 are AFM images of self-assembled C-duplex.

As shown in FIGS. 22 to 25 , the L-duplex formed a self-assembly with a highly dense structure, whereas the C-duplex formed a self-assembly with a loose and less connected structure. The linear or cyclic three-dimensional structure caused a difference in the formation of the double helix of L-duplex and C-duplex, which seems to have affected the self-assembly of L-duplex and C-duplex.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Self-Assembling Cyclic Peptide-Oligonucleotide Conjugates and Manufacturing Method Thereof <130> HPC9393 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> artificial sequence <220> <223> peptide <400> 1 Leu Leu Leu Phe Gln Gln Lys Lys 1 5 <210> 2 <211> 8 <212> PRT <213> artificial sequence <220> <223> peptide <400> 2 Lys Lys Gln Gln Phe Leu Leu Leu 1 5 <210> 3 <211> 8 <212> PRT <213> artificial sequence <220> <223> peptide <400> 3 Gln Gln Phe Leu Leu Leu Lys Lys 1 5 <210> 4 <211> 8 <212> PRT <213> artificial sequence <220> <223> peptide <400> 4 Lys Lys Leu Leu Leu Phe Gln Gln 1 5 <210> 5 <211> 5 <212> DNA <213> artificial sequence <220> <223> oligonucleotide <400> 5 gcgag 5 <210> 6 <211> 5 <212> DNA <213> artificial sequence <220> <223> oligonucleotide <400> 6 gaggc 5 <210> 7 <211> 5 <212> DNA <213> artificial sequence <220> <223> oligonucleotide <400> 7 aggcg 5 <210> 8 <211> 5 <212> DNA <213> artificial sequence <220> <223> oligonucleotide <400> 8 cgagg 5 <210> 9 <211> 5 <212> DNA <213> artificial sequence <220> <223> oligonucleotide <400> 9 ctcgc 5 <210> 10 <211> 5 <212> DNA <213> artificial sequence <220> <223> oligonucleotide <400> 10 tcgcc 5 <210> 11 <211> 5 <212> DNA <213> artificial sequence <220> <223> oligonucleotide <400> 11 cgcct 5 <210> 12 <211> 5 <212> DNA <213> artificial sequence <220> <223> oligonucleotide <400> 12 gcctc 5 <210> 13 <211> 5 <212> DNA <213> artificial sequence <220> <223> oligonucleotide <400> 13 cctcg 5

Claims (16)

Self-assembling cyclic peptide-oligonucleotide complex represented by Formula 1:
[Formula 1]

In Formula 1,
The Pep is a peptide represented by the amino acid sequence of SEQ ID NO: 1, and the oligo is an oligonucleotide represented by SEQ ID NO: 5 or 9. delete delete delete According to claim 1,
The self-assembling cyclic peptide-oligonucleotide complex, characterized in that the complex is represented by the following formula (4) or formula (5).
[Formula 4]

[Formula 5]

According to claim 1,
The cyclic peptide-oligonucleotide complex is a cyclic peptide-oligonucleotide complex, characterized in that it forms a self-assembly in solution . A self-assembly formed by self-assembling the self-assembling cyclic peptide-oligonucleotide complex according to claim 1 in a solution. According to claim 7,
The self-assembly is in the form of spherical micelles or vesicles with an average diameter of 10 to 30 nm. 1) preparing a linear peptide-oligonucleotide complex by mixing the peptide represented by Formula 6 and the oligonucleotide represented by Formula 7; and
2) cyclization by adding CuSO ₄ , ascorbic acid and butanol to the linear peptide-oligonucleotide complex;
[Formula 6]

[Formula 7]

The Pep is a peptide represented by the amino acid sequence of SEQ ID NO: 1, and the oligo is an oligonucleotide represented by SEQ ID NO: 5 or 9. According to claim 9,
The oligonucleotide represented by Formula 6 in step 1) is
1-a) treating the oligonucleotide represented by Formula 6 with a reducing agent; a method for producing a cyclic peptide-oligonucleotide complex, characterized in that disulfide bonds are removed from the 3' end. According to claim 9,
A method for producing a cyclic peptide-oligonucleotide complex, characterized in that the oligonucleotide represented by formula 6 and the peptide represented by formula 7 in step 1) are mixed in a molar ratio of 1: 1 to 4. According to claim 9,
In step 1), a method for preparing a cyclic peptide-oligonucleotide complex, characterized in that it further comprises an organic solvent. According to claim 12,
The organic solvent is ethanol, methanol, isopropyl alcohol, butanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile, dichloromethane, ethyl acetate, hexane, diethyl ether, A method for producing a cyclic peptide-oligonucleotide complex, characterized in that at least one organic solvent selected from the group consisting of benzene, chloroform and acetone. According to claim 9,
In step 2),
A method for producing a cyclic peptide-oligonucleotide complex, characterized in that the equivalent ratio of the linear peptide-oligonucleotide complex, CuSO ₄ and ascorbic acid is 1: 2-4: 5-7. According to claim 9,
Step 2) is a method for producing a cyclic peptide-oligonucleotide complex, characterized in that carried out under an inert atmosphere. A self-assembling cyclic peptide-oligonucleotide complex represented by Formula 1 prepared according to the method of claim 9:
[Formula 1]

In Formula 1,
The Pep is a peptide represented by the amino acid sequence of SEQ ID NO: 1, and the oligo is an oligonucleotide represented by SEQ ID NO: 5 or 9.

Images